The overall objective of this proposal is to define the structural details of the interaction of antithrombin with the multi-ligand low density lipoprotein receptor-related protein (LRP). The scope of this project includes the definition of the discrete sites of interaction within LRP for one of its ligands, antithrombin; the sites within antithrombin for interaction with LRP; and the sites within the LRP intracellular chaperone RAP that inhibit the binding of antithrombin to LRP. In Specific Aim 1, the site on antithrombin responsible for interaction with LRP will be determined using synthetic peptides and peptide antibodies as binding antagonists, and site-specific mutagenesis of antithrombin to locate specific participating amino acid residues. In addition, the role of heparan sulfate proteoglycans (HSPG) on the cell surface in antithrombin:LRP binding will be investigated. In Specific Aim 2, the site on LRP responsible for its interaction with antithrombin will be determined using LRP minireceptors that represent three different complement-type ligand binding clusters. After establishing this regional binding site, the precise LRP binding site for antithrombin will be identified using site-specific mutagenesis. In addition, the minireceptor that binds to antithrombin will be expressed in large amounts in bacteria, refolded externally and used for X-ray crystallographic analysis as well as for other structural and functional studies. In Specific Aim 3, the site on RAP that inhibits the binding of antithrombin to LRP will be determined in a similar fashion. This will include definition of the heparin binding site within the third repeat of RAP that may be involved in RAP's multiple LRP interactions, as well as other regions of RAP that may be generally involved in inhibiting ligand binding to LRP and/or uniquely involved in inhibiting antithrombin binding.